Ceramide is being recognized by many as an important player in the process of apoptosis including steps mediated by mitochondria. It can be produced locally by mitochondrial ceramide synthase and has been shown to induce the release of cytochrome c. This proposal presents evidence that ceramides can form channels in phospholipid membranes without the aid of proteins. These channels appear to be large enough to account for the efflux of cytochrome c and other pro-apoptotic factors from the mitochondrial intermembrane space resulting in the activation of the caspase pathway and thus initiating full-flown apoptosis. The focus of the proposal is to test the ability of short and long-chain ceramides and related lipids to form channels large enough and stable enough to allow this protein release. The properties of any channels formed will be studied electrophysiologically with planar phospholipid membranes and by performing flux experiments on liposomes and with isolated mitochondria. Factors will be investigated which may stabilize or destabilize these channels. The biologically inactive, dihydroceramide, and other ceramide analogues varying in their ability to induce apoptosis will be used to correlate apoptosis-inducing ability with channel-forming ability. These will also be examined for their influence on channel formation by ceramides and related lipids. The use of ceramide analogues will also identify key structural features important in channel formation. Predictions from the working model of ceramide channel structure will be tested. Possible interactions between these lipids and members of the Bcl-2 family of proteins that up and down regulate apoptosis will be investigated. Electron microscopy will be used to visualize these structures in replicas of freeze-fractured liposomes. Experiments will also be aimed at understanding why plasma membranes are resistant to the formation of ceramide channels. It is hoped that these insights will yield targets for the design of drugs that may inhibit or stimulate ceramide-mediated apoptosis. These might prove useful controlling the apoptotic process and thus treat diseases such as cancer, heart disease and stroke.